Whole genome shotgun sequencing, assembly and finishing is typically the strategy of choice for microbial and fungal genome sequencing. The cost-advantages and simplicity of the whole genome approach relative to a BAC based or hybrid sequencing strategy argue strongly for its continued development and application in future sequencing projects. However, a major problem with the BAC-based approaches is the high cost and operational burden associated with the production of 15,000-25,000 individual BAC subclone libraries, the 15-20% waste associated with re-sequencing the vector, as well as the unavoidable E. coli contamination, the need to deal with transposon and bacteriophage insertions, and the 20-50% waste in redundant sequencing of BAC overlaps. Although these costs can be reduced by sequencing the BACs at low coverage (using a hybrid BAC/WGS strategy, for example) or by using a pooling strategy, they cannot be eliminated. The need to generate a physical map by using restriction digest fingerprinting or by complex pooling and sequence based mapping strategies adds additional cost and operational overhead.
Thus, a need exists for more cost-efficient sequencing methods and for better methods of generating a reliable sequence-derived scaffold that can support the accurate selection of clones to finish any desired region of the genome with reduced operational burden, increased efficiency, elimination of problems associated with transposon and bacteriophage insertions and reduction in wasted time, effort and expense spent in redundant sequencing. Additionally, a need exists to provide a reliable and efficient method that facilitates whole genome assembly and/or karyotyping of a genome, and which enables the reliable and efficient detection of sequence inversion in a genome.
Current bacterial and yeast two-hybrid screening methods are useful to discover the identity of two interacting proteins. However, these methods suffer from the need for large numbers of transformations to be performed: one for each bait to be analyzed against one or more prey molecule. Although some methods have been developed to permit pools of ten to one hundred baits to be screened in parallel, these methods require additional handling steps to deconvolute the identities of the individual baits. Therefore, these methods only incrementally increase the efficiency of conventional two-hybrid systems.
Thus, a need exists for a method to increase the efficiency of two-hybrid systems for the identification of two interacting proteins.